Project Summary Synapses are fundamental to nervous system function and information processing in the normal and pathological brain. They are highly dynamic structures capable of supporting high firing rates and displaying a broad range of plasticity. A detailed picture of the molecular interactions occurring within a synapse is required to understand how synaptic protein dynamics ultimately shape activity in the nervous system in health and disease. In this proposal, we will investigate Munc13 and complexin (CPX), both highly conserved molecules that are crucial for proper synaptic function. Mice lacking complexin die at birth, and loss of a single isoform is associated with profound locomotor, sensory, and behavioral deficits. A human Cpx1 point mutation is associated with severe intellectual disability and seizures, and CPX expression is altered in a host of psychiatric and neurodegenerative diseases including Huntington?s, Parkinson?s, and Alzheimer?s disease. Although it is well-established that complexin plays a major role in synaptic function, its mode of action is controversial: there is evidence for both facilitatory and inhibitory roles of this protein in the regulation of synaptic transmission. Munc13 is a large synaptic hub protein that coordinates several proteins involved in synaptic vesicle fusion, and human mutations are associated with ALS, fatal myasthenia, microcephaly, and severe autism. We propose to study the molecular mechanisms underlying Munc13 and CPX function at the synapse using a unique and powerful combination of in vivo and in vitro approaches including physiology, quantitative imaging, behavioral assays, genetics, and protein/lipid biochemistry. We have developed innovative methods of investigating protein interactions in vivo by monitoring the dynamics of proteins exchanging between neighboring synapses using photoactivatable GFP. We will deploy these methods in C. elegans as a model system in which to study the protein interactions of CPX as well as domains of Munc13 and their binding partners in a functional synapse. By mutating either the pGFP-tagged protein or its binding partners, we have established that affinity changes lead to mobility changes. We will also elucidate the structure of CPX and Munc13 protein domains that mediate critical membrane interactions using a combination of biochemical and spectroscopic techniques. Detailed structure-function analyses will be conducted on a domain of Munc13 recently discovered in our lab. The experiments proposed here will provide new insights into the mechanisms that control neurotransmitter release, its modulation, and use-dependent plasticity in the brain.